Project Summary/Abstract Protein misfolding is known to play a central role in numerous neurodegenerative diseases such as Alzheimer's, amyotrophic lateral sclerosis (ALS), Huntington's, Parkinson's and transmissible spongiform encephalopathies such as Creutztfeld-Jakob's disease. In ALS, the misfolded structural state of copper-zinc super-oxide dismutase (SOD1) imparts a function to this normally beneficial protein that is essential to disease pathophysiology. However, the exact nature and mechanism(s) of this acquired function have remained elusive despite years of intense research. Most research has proposed that the deposition of aggregates of amyloid- like SOD1 protein directly affects neurological function. Based on our preliminary studies, our overall hypothesis is that these misfolded SOD1 proteins form "toxic ion channels", which in turn create ionic imbalances (such as calcium overload) that result in neurotoxicity. Aim1: To test the hypothesis that a mutant and perhaps, under certain conditions, wild-type SOD1 protein co- localize to subcellular lipid membrane domains especially the cytoplasmic face of the outer mitochondrial membrane. Aim2: To test the hypothesis that the ultrastructure of mutant and perhaps wild-type SOD1 undergoes conformational changes in "toxic channels". Aim 3: To test the hypothesis that nano-scale intermolecular forces and oligomeric states govern the insertion and stabilization of mutant and perhaps wild- type SOD1 molecules in lipid membrane. Aim 4: To test the hypothesis that mutant SOD1 and perhaps wild- type SOD1, under certain conditions, render the lipid membrane permeable by certain ions. Our research design and methods are to: (Aim1) Use TIRF, FRET and immuno-fluorescence microscopy techniques to co-localize the "toxic channels" to cellular membranes and also generate a synthetic antibody against "toxic channels". (Aim2) Use high resolution, fluid atomic force microscopy (fAFM) and (Aim 3) fAFM force measurements to determine the subunit structure, conformational changes and the intermolecular forces that stabilize "toxic channels". And (Aim 4) use single-channel electrophysiology recordings to measure and modulate ion flow through the "toxic channels" in order to understand channel properties and characteristics. Today tens of millions of individuals suffer the debilitating and fatal symptoms of neurodegenerative diseases (ND). For the vast majority of these individuals, no effective treatment exists currently. We have proposed a new model describing certain forms of ND as resulting from "toxic ion channels" which invade host neuronal cell membranes and give rise to disease pathophysiology. Since it is new, the "toxic channel" model has not yet gained wide acceptance or significant research funding. However, presented in this proposal is new and compelling experimental evidence (regarding ALS) which supports the "toxic channel" model. If our model is correct, the proposed work will be putting us on the right track to finding new effective treatments for ND and other protein misfolding diseases involving "toxic channels". With successful funding we are convinced that continuation of our work will lead to the development of new antibody-based diagnostics, as well as effective new therapeutic treatments for ALS.